The invention relates to apoptosis in cells, particularly cell involved in fertility.
Apoptosis is a fundamental process of cell death required for the elimination of unwanted cells in multicellular organisms and involves an ordered cascade of events leading to hallmark morphological changes including nuclear condensation, chromosome laddering, and membrane blebbing. In one specific example, apoptosis plays a prominent role during all stages of sperm development. Spermatogenesis is a process that results in the generation of mature sperm cells from primary germ cells, and some of the events affected by apoptosis include the elimination of unwanted cells and the prevention of the death of those cells destined to become functional sperm (Hsueh et al., Recent Prog. Horm. Res. 51: 433, 1996; Furuchi et al., Development 122: 1703, 1996).
Much of the current knowledge of the biochemical pathways involved in apoptosis comes from the study of viruses. The baculoviral proteins involved in apoptosis, CpIAP and OpIAP, are characterized by two amino terminal cys/his motifs (Xaa3xe2x80x94Rxe2x80x94Xaa20-23xe2x80x94Gxe2x80x94Xaa11xe2x80x94Cxe2x80x94Xaa2xe2x80x94Cxe2x80x94Xaa16xe2x80x94Hxe2x80x94Xaa6xe2x80x94Cxe2x80x94Xaa3) (SEQ ID NOs: 6-9) and a carboxy terminal Cxe2x80x94Xaa2xe2x80x94Cxe2x80x94Xaa11xe2x80x94Cxe2x80x94Xaaxe2x80x94Hxe2x80x94Xaa3xe2x80x94Cxe2x80x94Xaa2xe2x80x94Cxe2x80x94Xaa6xe2x80x94Cxe2x80x94Xaa2xe2x80x94C RING zinc finger motif (SEQ ID NO: 10) reviewed in Clem et al., Cell Death Differ. 3: 9, 1996). The two amino terminal motifs, termed Baculovirus IAP Repeat (BIR) domains, are the hallmark of the IAP family of proteins and are present as two or three copies in all IAP members discovered to date.
The first mammalian IAP (inhibitor of apoptosis protein), NAIP, was identified during a positional cloning effort seeking candidate genes for spinal muscular atrophy (Roy et al., Cell 80: 167, 1995). Following the identification of NAIP, three other human IAPs, HIAP-1, HIAP-2, and XIAP (Liston et al., Nature 379: 349, 1996), and their murine homologues MIAP-1, MIAP-2, and MIAP-3 (Farahani et al., Genomics 42: 514, 1997), have been reported. In addition, two Drosophila IAPs, DIAP-1 and DIAP-2, and one chicken IAP, ITA have been described (see Liston et al., Apoptosis 2: 423, 1997). A structural comparison of these IAPs is shown in FIG. 1.
The physiological role of XIAP remains elusive. Recent work has demonstrated that XIAP directly inhibits two of the caspases, namely caspase-3 (also known as CPP32, Apopain, or YAMA) and caspase-7 (Devereaux et al., Nature 388: 300, 1997).
In general, the invention features methods and reagents useful for the treatment of excessive or insufficient apoptosis, particularly in testicular cells. The methods and reagents of the invention are useful in diagnosing and treating testicular cancers, cancers in non-testicular tissues, male infertility, and for achieving male birth control.
In a first aspect, the invention features a substantially pure nucleic acid molecule encoding a TIAP polypeptide. In one embodiment, the nucleic acid molecule has a sequence that is substantially identical to SEQ ID NO: 1. In another embodiment, the nucleic acid molecule has a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1. In other embodiments, the TIAP polypeptide has a sequence that is substantially identical to SEQ ID NO: 2, or has a sequence that is at least 80% identical to SEQ ID NO: 2. In a preferred embodiment, the TIAP has the amino acid sequence of SEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1. Preferably, identity is measured using sequence software analysis, such as the Sequence Analysis Software Package of the Genetics Computer Group. TIAP polypeptides encoded by the aforementioned nucleic acids are another aspect of the invention.
In a second aspect, the invention features a substantially pure nucleic acid molecule corresponding to at least fifteen nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, where the nucleic acid molecule is an antisense nucleic acid molecule that is sufficient to decrease TIAP biological activity. In various embodiments, the antisense nucleic acid molecule corresponds to: at least thirty nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, at least fifty nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, or at least 100 nucleotides of a nucleic acid molecule encoding a TIAP polypeptide. In other preferred embodiments, the TIAP biological activity is decreased by at least 20%, at least 40%, at least 60%, or at least 80%. In yet another embodiment of the second aspect of the invention, the antisense nucleic acid molecule is in a vector that is capable of directing expression of the antisense nucleic acid molecule in a vector-containing cell.
In a third aspect, the invention features a vector including a substantially pure nucleic acid molecule encoding a TIAP polypeptide, the vector being capable of directing expression of the polypeptide in a vector-containing cell.
In a fourth aspect, the invention features a cell that contains a substantially pure nucleic acid molecule encoding a TIAP polypeptide. In one preferred embodiment, the nucleic acid molecule is expressed in the cell. In another embodiment, the cell is selected from a cell from the testis. In yet another embodiment, the cell is present in an animal having a condition that is associated with excessive or insufficient cell death.
In a fifth aspect, the invention features a transgenic animal generated from a cell genetically engineered to lack a nucleic acid molecule encoding a TIAP polypeptide, where the transgenic animal lacks expression of the TIAP polypeptide.
In a sixth aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid molecule replacing DNA encoding a TIAP polypeptide, where the nucleic acid molecule is expressed in the transgenic animal.
In one embodiment of the first six aspects of the invention, the nucleic acid molecule is from an animal. In another embodiment, the nucleic acid molecule is genomic DNA or cDNA. In a preferred embodiment of the first six aspects of the invention, the nucleic acid molecule is operably linked to regulatory sequences for expression of the polypeptide and where the regulatory sequences include a promoter. Such a promoter may be from the TIAP gene, or may be selected from the group consisting of a constitutive promoter, a promoter that is inducible by one or more external agents, and a cell-type specific promoter.
In a seventh aspect, the invention features a method of identifying a compound that modulates TIAP biological activity that includes: (a) providing a cell including a TIAP gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the TIAP gene, where an alteration in the expression in response to the candidate compound relative to an cell not contacted with the candidate compound indicates a compound that modulates TIAP biological activity. In one embodiment of this aspect of the invention, the cell is transformed.
In an eighth aspect, the invention features a method of identifying a compound that modulates TIAP biological activity that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a TIAP gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where an alteration in the expression in response to the candidate compound relative to an cell not contacted with the candidate compound indicates a compound that modulates TIAP biological activity. In one embodiment of this aspect of the invention, the cell is transformed.
In a ninth aspect, the invention features a method of identifying a compound that is able to modulate TIAP biological activity in a cell that includes the steps of: (a) providing a expressing a normal amount of TIAP; (b) contacting the cell with a candidate compound; and (c) measuring level of apoptosis in the cell, where an alteration in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that modulates TIAP biological activity. Preferably, the cell is a germ-line cell.
In one embodiment of the seventh, eighth, and ninth aspect of the invention, the alteration that is an increase indicates the compound is inhibiting TIAP biological activity, and the alteration that is a decrease indicates the compound is increasing TIAP biological activity. In other embodiments, the TIAP is from an animal.
In a tenth aspect, the invention features a method of increasing apoptosis in a cell that includes administering to the cell an apoptosis-inducing amount of a TIAP polypeptide or fragment thereof. Preferably, the cell is a germ-line cell.
In an eleventh aspect, the invention features a method of increasing apoptosis in a cell, the method including administering to the cell a compound which modulates TIAP biological activity. Such a compound may be selected from a group consisting of a neutralizing antibody that specifically binds to TIAP, a polypeptide fragment of a TIAP polypeptide, a mutant of a TIAP polypeptide, a TIAP antisense nucleic acid molecule, and a nucleic acid molecule encoding a TIAP polypeptide, a mutant thereof, or a polypeptide fragment thereof. Preferably, the cell is a germ-line cell.
In one embodiment of the tenth and eleventh aspects of the invention, the cell is in an animal. In another embodiment, the TIAP is from an animal. In preferred embodiments, the animal is desired to have reduced fertility, is diagnosed with cancer, or has a predisposition to develop cancer, for example, a cancer that is selected from a group consisting of testicular cancer, scrotal cancer, prostate cancer, and a cancer in which aberrant expression of TIAP is observed.
In a twelfth aspect, the invention features a method of increasing fertility in an animal that includes providing a transgene encoding a TIAP polypeptide or fragment thereof to a cell of the animal, the transgene being positioned for expression in the cell. Preferably, the cell is a germ-line cell.
In a thirteenth aspect, the invention features a method of increasing fertility in an animal that includes administering to a cell of the animal an excessive amount of TIAP polypeptide or fragment thereof. Preferably, the cell is a germ-line cell.
In a fourteenth aspect, the invention features a method of increasing fertility in an animal that includes administering to a cell of the animal a compound which modulates TIAP biological activity. Preferably, the cell is a germ-line cell. Such a compound may be selected from the group consisting of a full-length TIAP polypeptide, a fragment of a TIAP polypeptide, and a nucleic acid molecule encoding a TIAP polypeptide.
In various embodiments of the twelfth, thirteenth, and fourteenth aspects of the invention, the animal is diagnosed as having a condition involving excessive apoptosis, for example, a condition that is associated with reduced fertility.
In a fifteenth aspect, the invention features a method of diagnosing an animal for the presence of disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis. Preferably the apoptosis is in a germ-line cell. The method includes isolating a sample of a nucleic acid molecule from the animal and determining whether the nucleic acid molecule includes a TIAP mutation, the mutation being an indication that the animal has an apoptosis disease or an increased likelihood of developing a disease involving apoptosis in a cell (e.g., a germ-line cell).
In a sixteenth aspect, the invention features a second method of diagnosing an animal for the presence of a disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis. Preferably the apoptosis is in a germ-line cell. The second method includes measuring TIAP gene expression in a sample from the animal, an alteration in the expression relative to a sample from an unaffected animal being an indication that the animal has an apoptosis disease or increased likelihood of developing an apoptosis disease in a cell (e.g., a germ-line cell). In a preferred embodiment of this aspect of the invention, the gene expression is measured by assaying the amount of TIAP polypeptide or TIAP biological activity in the sample. In another embodiment of this aspect of the invention, the TIAP polypeptide is measured by immunological methods or by assaying the amount of TIAP RNA in the sample.
In a seventeenth aspect, the invention features a kit for diagnosing an animal for the presence of a disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis, where the kit includes a substantially pure antibody that specifically binds a TIAP polypeptide.
In an eighteenth aspect, the invention features kit for diagnosing an animal for the presence of a disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis, where the kit includes a material for measuring TIAP RNA.
In a nineteenth aspect, the invention features a kit for diagnosing an animal for the presence of a disease involving altered apoptosis or an increased likelihood of developing a disease involving altered apoptosis, where the kit includes: (a) a substantially pure antibody that specifically binds a TIAP polypeptide; or (b) a material for measuring TIAP RNA. Preferably, the kit has both the antibody and the material for measuring TIAP RNA. In a preferred embodiment of this aspect of the invention, the kit further includes a means for detecting the binding of the antibody to the TIAP polypeptide. In another embodiment, the material for measuring TIAP RNA is a nucleic acid probe.
In a twentieth aspect, the invention features a method of obtaining a TIAP polypeptide that includes: (a) providing a cell with a nucleic acid molecule encoding a TIAP polypeptide, the nucleic acid molecule being positioned for expression in the cell; (b) culturing the cell under conditions for expressing the nucleic acid molecule; and (c) isolating the TIAP polypeptide. In one embodiment of this aspect of the invention, the nucleic acid molecule further includes a promoter that is inducible by one or more external agents.
In a twenty-first aspect, the invention features a method of isolating a TIAP gene or portion thereof having sequence identity to human TIAP that includes amplifying by polymerase chain reaction the TIAP gene or portion thereof using oligonucleotide primers where the primers: (a) are each greater than 13 nucleotides in length; (b) each have regions of complementarity to opposite DNA strands in a region of the nucleotide sequence of SEQ ID NO: 1; and (c) optionally contain sequences capable of producing restriction endonuclease cut sites in the amplified product; and isolating the TIAP gene or portion thereof.
In a twenty-second aspect, the invention features a method of isolating a TIAP gene or fragment thereof from a cell that includes: (a) providing a sample of cellular nucleic acid; (b) providing a pair of oligonucleotides having sequence identity to a conserved region of a TIAP gene; (c) combining the pair of oligonucleotides with the cellular nucleic acid sample under conditions suitable for polymerase chain reaction-mediated nucleic acid amplification; and (d) isolating the amplified TIAP gene or fragment thereof.
In a preferred embodiment of the twentieth, twenty-first, and twenty-second aspects of the invention, the polymerase chain reaction is carried out using a reverse-transcription polymerase chain reaction. In another embodiment, the reverse-transcription polymerase chain reaction is RACE.
In a twenty-third aspect, the invention features a method of identifying a TIAP gene in a cell from an animal that includes: (a) providing a sample of cellular nucleic acid from the animal; (b) providing a detectably-labeled nucleic acid sequence having identity to a conserved region of a known TIAP gene; (c) contacting the sample with the detectably-labeled nucleic acid sequence under hybridization conditions that provide detection of genes having 50% or greater nucleotide sequence identity to the detectably labeled nucleic acid sequence; and (d) identifying the TIAP gene. Preferably, the nucleic acid sequence includes at least a portion of SEQ ID NO: 1.
In a twenty-fourth aspect, the invention features a method of identifying a TIAP gene that includes: (a) providing a cell sample from an animal; (b) introducing by transformation into the cell sample a candidate TIAP gene; (c) expressing the candidate TIAP gene within the cell sample; and (d) determining whether the sample exhibits an altered level of apoptosis whereby an alteration in the level of apoptosis identifies a TIAP gene. In one embodiment of this aspect of the invention, the cell sample is selected from the group consisting of a spermatogonium, a spermatocyte, a spermatid, a sperm cell, a Sertoli cell, a fibroblast, a neuron, a myocardial cell, and an embryonic stem cell.
In a twenty-fifth aspect, the invention features a TIAP polypeptide. The polypeptide may be used in modulating apoptosis in a cell. Preferably, the cell is a germ-line cell.
In a twenty-sixth aspect, the invention features a substantially pure antibody that specifically binds a TIAP polypeptide, or a fragment or a mutant thereof. In one embodiment of this aspect of the invention, the TIAP polypeptide is from an animal.
In twenty-seventh aspect, the invention features a substantially pure nucleic acid molecule operably linked to a nucleic acid sequence encoding a TIAP polypeptide. In one embodiment, nucleic acid molecule is a TIAP gene promoter. In another embodiment, the nucleic acid molecule has a nucleic acid sequence that is substantially identical to a sequence found within SEQ ID NO: 5.
In a twenty-eighth aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid molecule operably linked to a TIAP gene promoter. In one embodiment of this aspect of the invention, the TIAP gene promoter has a nucleic acid sequence that is substantially identical to a sequence found within SEQ ID NO: 5. In another embodiment, the nucleic acid molecule encodes a toxin, for example, diptheria toxin or ricin.
By xe2x80x9cTIAPxe2x80x9d is meant a protein or polypeptide which is a member of the IAP family of proteins and which is encoded by a nucleic acid molecule having a high degree of sequence identity to the upper nucleic acid sequence shown in FIG. 4A (SEQ ID NO: 1). Preferably, the sequence encoding TIAP is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identical to SEQ ID NO: 1. Preferably, identity is measured using sequence software analysis, such as the Sequence Analysis Software Package of the Genetics Computer Group.
By xe2x80x9cgerm-line cellxe2x80x9d is meant a cell, progenitor, or progeny thereof, which is a product of a meiotic cell division. Preferably, the germ-line cell of the invention is a male germ-line cell and resides in the testis.
By xe2x80x9cmale birth controlxe2x80x9d is meant a composition which, when administered to a male animal, reduces the sperm count in the administered animal relative to the same animal not administered the male birth control composition. Preferably, the reduction in sperm count is at least 10%, preferably 20%, more preferably at least 50%, and most preferably, at least 80%, as compared to an animal not administered the male birth control.
By xe2x80x9cBIR domainxe2x80x9d is meant a domain having the amino acid sequence of the consensus sequence: Xaa3xe2x80x94Argxe2x80x94Xaa20-23xe2x80x94Glyxe2x80x94Xaa11xe2x80x94Cysxe2x80x94Xaa2xe2x80x94Cysxe2x80x94Xaa16xe2x80x94Hisxe2x80x94Xaa6xe2x80x94Cysxe2x80x94Xaa3, wherein Xaa is any amino acid residue (SEQ ID NOs: 6-9). Preferably, the sequence is substantially identical to one of the BIR domain sequences provided for XIAP or TIAP described herein.
By xe2x80x9cring zinc fingerxe2x80x9d or xe2x80x9cRZFxe2x80x9d is meant a domain having the amino acid sequence of the consensus sequence: Cxe2x80x94Xaa2xe2x80x94Cxe2x80x94Xaa11xe2x80x94Cxe2x80x94Xaaxe2x80x94Hxe2x80x94Xaa3xe2x80x94Cxe2x80x94Xaa2xe2x80x94Cxe2x80x94Xaa6xe2x80x94Cxe2x80x94Xaa2xe2x80x94C, where Xaa is any amino acid (SEQ ID NO: 10). Preferably, the sequence is substantially identical to the RZF domains provided herein for XIAP or TIAP.
By xe2x80x9cmodulating apoptosisxe2x80x9d or xe2x80x9caltering apoptosisxe2x80x9d is meant increasing or decreasing the number of cells which undergo apoptosis in a given cell population. Preferably, the cell population is selected from a group including spermatogonium, spermatocytes, epithelial cells, fibroblasts, or any other cell line known to undergo apoptosis in a laboratory setting (e.g., the baculovirus infected insect cells). It will be appreciated that the degree of modulation provided by a TIAP or modulating compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis which identifies a TIAP or a compound which modulates a TIAP.
By xe2x80x9cinhibiting apoptosisxe2x80x9d is meant any decrease in the number of cells which undergo apoptosis relative to an untreated control. Preferably, the decrease is at least 25%, more preferably the decrease is 50%, and most preferably the decrease is at least one-fold.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, polypeptide. A substantially pure TIAP polypeptide may be obtained, for example, by extraction from a natural source (e.g., testis); by expression of a recombinant nucleic acid encoding a TIAP polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure nucleic acid moleculexe2x80x9d is meant nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant nucleic acid molecule which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR amplification or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a TIAP-specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody which recognizes and binds a protein but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes protein.
By xe2x80x9ctoxinxe2x80x9d is meant a protein or polypeptide which kills the cell in which it is expressed. Exemplary toxins include, without limitation, diptheria toxin, cholera toxin, and ricin.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents, such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules into a cell. Lipofection, calcium phosphate precipitation, retroviral delivery, electroporation and biolistic transformation are just a few of the teachings which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a TIAP polypeptide.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a TIAP polypeptide, a recombinant protein or a RNA molecule).
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant hybridization in 2xc3x97 SSC at 40xc2x0 C. with a nucleic acid probe length of at least 30 nucleotides. For other definitions of high stringency conditions, see Ausubel et al., 1994, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 6.3.1-6.3.6, hereby incorporated by reference.
By xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, xcex2-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and xcex2-galactosidase.
By xe2x80x9cpromoterxe2x80x9d is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cdetectably-labelledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g., with an isotope such as 32P or 35S) and nonradioactive labelling (e.g., chemiluminescent labelling, e.g., fluorescein labelling).
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a TIAP gene. The preferred antisense nucleic acid molecule is one which is capable of lowering the level of polypeptide encoded by the complementary gene when both are expressed in a cell. Preferably, the polypeptide level is lowered by at least 10%, more preferably at least 25%, and most preferably, at least 50%, as compared to the polypeptide level in a cell expressing only the gene, and not the complementary antisense nucleic acid molecule.